Tumor suppressor protein CYLD regulates morphogenesis of dendrites and spines.

Cylindromatosis tumor suppressor protein (CYLD) was initially identified as a tumor suppressor deubiquitylating protein in familial cylindromatosis patients. Proteomic analyses using rodent brain samples revealed enrichment of CYLD in purified postsynaptic density fractions. Here, we report that CYLD regulates dendritic growth and postsynaptic differentiation in mouse hippocampal neurons. CYLD showed diffuse localization in rapidly growing dendrites, but was gradually concentrated in spines. Overexpression and knockdown of CYLD in the early stage of cultured neurons demonstrated that CYLD positively regulated dendritic growth. Phenotypes in dendritic morphogenesis induced by CYLD overexpression and knockdown could be reversed by manipulation of the critical acetylation site of α-tubulin, suggesting tubulin acetylation is a downstream pathway of CYLD-dependent dendritic growth. Overexpression and knockdown of CYLD in the later stage of cultured neurons revealed that CYLD promoted formation of postsynaptic spines. Influence of CYLD on spines was not affected by co-expression of acetylation mutant forms of α-tubulin, indicating that CYLD regulates dendritic growth and spine formation through different molecular mechanisms. Analyses with the truncated and mutated forms of CYLD demonstrated that the first microtubule-binding domain of CYLD was critical for spine formation. These results suggest important roles of CYLD in sequential promotion of dendritic growth and postsynaptic spine maturation.

Structural dynamics of dendritic spines: molecular composition, geometry and functional regulation.

The development of dendritic spines with specific geometry and membrane composition is critical for proper synaptic function. Specific spine membrane architecture, sub-spine microdomains and spine head and neck geometry allow for well-coordinated and compartmentalized signaling, disruption of which could lead to various neurological diseases. Research from neuronal cell culture, brain slices and direct in vivo imaging indicates that dendritic spine development is a dynamic process which includes transition from small dendritic filopodia through a series of structural refinements to elaborate spines of various morphologies. Despite intensive research, the precise coordination of this morphological transition, the changes in molecular composition, and the relation of spines of various morphologies to function remain a central enigma in the development of functional neuronal circuits. Here, we review research so far and aim to provide insight into the key events that drive structural change during transition from immature filopodia to fully functional spines and the relevance of spine geometry to function.

Predicting capillary vessel network hemodynamics in silico by machine learning.

Blood velocity and red blood cell (RBC) distribution profiles in a capillary vessel cross-section in the microcirculation are generally complex and do not follow Poiseuille's parabolic or uniform pattern. Existing imaging techniques used to map large microvascular networks in vivo do not allow a direct measurement of full 3D velocity and RBC concentration profiles, although such information is needed for accurate evaluation of the physiological variables, such as the wall shear stress (WSS) and near-wall cell-free layer (CFL), that play critical roles in blood flow regulation, disease progression, angiogenesis, and hemostasis. Theoretical network flow models, often used for hemodynamic predictions in experimentally acquired images of the microvascular network, cannot provide the full 3D profiles either. In contrast, such information can be readily obtained from high-fidelity computational models that treat blood as a suspension of deformable RBCs. These models, however, are computationally expensive and not feasible for extension to the microvascular network at large spatial scales up to an organ level. To overcome such limitations, here we present machine learning (ML) models that bypass such expensive computations but provide highly accurate and full 3D profiles of the blood velocity, RBC concentration, WSS, and CFL in every vessel in the microvascular network. The ML models, which are based on artificial neural networks and convolution-based U-net models, predict hemodynamic quantities that compare very well against the true data but reduce the prediction time by several orders. This study therefore paves the way for ML to make detailed and accurate hemodynamic predictions in spatially large microvascular networks at an organ-scale.

A computational study of red blood cell deformability effect on hemodynamic alteration in capillary vessel networks.

Capillary blood vessels, the smallest vessels in the body, form an intricate network with constantly bifurcating, merging and winding vessels. Red blood cells (RBCs) must navigate through such complex microvascular networks in order to maintain tissue perfusion and oxygenation. Normal, healthy RBCs are extremely deformable and able to easily flow through narrow vessels. However, RBC deformability is reduced in many pathological conditions and during blood storage. The influence of reduced cell deformability on microvascular hemodynamics is not well established. Here we use a high-fidelity, 3D computational model of blood flow that retains exact geometric details of physiologically realistic microvascular networks, and deformation of every one of nearly a thousand RBCs flowing through the networks. We predict that reduced RBC deformability alters RBC trafficking with significant and heterogeneous changes in hematocrit. We quantify such changes along with RBC partitioning and lingering at vascular bifurcations, perfusion and vascular resistance, and wall shear stress. We elucidate the cellular-scale mechanisms that cause such changes. We show that such changes arise primarily due to the altered RBC dynamics at vascular bifurcations, as well as cross-stream migration. Less deformable cells tend to linger less at majority of bifurcations increasing the fraction of RBCs entering the higher flow branches. Changes in vascular resistance also seen to be heterogeneous and correlate with hematocrit changes. Furthermore, alteration in RBC dynamics is shown to cause localized changes in wall shear stress within vessels and near vascular bifurcations. Such heterogeneous and focal changes in hemodynamics may be the cause of morphological abnormalities in capillary vessel networks as observed in several diseases.

Application of machine learning in predicting blood flow and red cell distribution in capillary vessel networks.

Capillary blood vessels in the body partake in the exchange of gas and nutrients with tissues. They are interconnected via multiple vascular junctions forming the microvascular network. Distributions of blood flow and red cells (RBCs) in such networks are spatially uneven and vary in time. Since they dictate the pathophysiology of tissues, their knowledge is important. Theoretical models used to obtain flow and RBC distribution in large networks have limitations as they treat each vessel as a one-dimensional segment and do not explicitly consider cell-cell and cell-vessel interactions. High-fidelity computational models that accurately model each individual RBC are computationally too expensive to predict haemodynamics in large vascular networks and over a long time. Here we investigate the applicability of machine learning (ML) techniques to predict blood flow and RBC distributions in physiologically realistic vascular networks. We acquire data from high-fidelity simulations of deformable RBC suspension flowing in the networks. With the flow and haematocrit specified at an inlet of vasculature, the ML models predict the time-averaged flow rate and RBC distributions in the entire network, time-dependent flow rate and haematocrit in each vessel and vascular bifurcation in isolation over a long time, and finally, simultaneous spatially and temporally evolving quantities through the vessel hierarchy in the networks.

Investigation of Signals and Transcription Factors for The Generation of Female Germ-Like Cells.

Objective: Primordial germ cell (PGCs) lines are a source of a highly specialized type of cells, characteristically oocytes,
during female germline development in vivo. The oocyte growth begins in the transition from the primary follicle. It is
associated with dynamic changes in gene expression, but the gene-regulating signals and transcription factors that control oocyte growth remain unknown. We aim to investigate the differentiation potential of mouse bone marrow mesenchymal stem cells (mMSCs) into female germ-like cells by testing several signals and transcription factors.
Materials and Methods: In this experimental study, mMSCs were extracted from mice femur bone using the flushing
technique. The cluster-differentiation (CD) of superficial mesenchymal markers was determined with flow cytometric analysis. We applied a set of transcription factors including retinoic acid (RA), titanium nanotubes (TNTs), and fibrin such as TNT-coated fibrin (F+TNT) formation and (RA+F+TNT) induction, and investigated the changes in gene, MVH/ DDX4, expression and functional screening using an in vitro mouse oocyte development condition. Germ cell markers expression, (MVH / DDX4), was analyzed with Immunocytochemistry staining, quantitative transcription-polymerase chain reaction (RT-qPCR) analysis, and Western blots.
Results: The expression of CD was confirmed by flow cytometry. The phase determination of the TNTs and F+TNT were confirmed using x-ray diffraction (XRD) and scanning electron microscope (SEM), respectively. Remarkably, applying these transcription factors quickly induced pluripotent stem cells into oocyte-like cells that were sufficient to generate female germlike cells, growth, and maturation from mMSCs differentiation. These transcription factors formed oocyte-like cells specification of stem cells, epigenetic reprogramming, or meiosis and indicate that oocyte meiosis initiation and oocyte growth are not separable from the previous epigenetic reprogramming in stem cells in vitro.
Conclusion: Results suggested several transcription factors may apply for arranging oocyte-like cell growth and supplies an alternative source of in vitro maturation (IVM).

Polo kinase interacts with RacGAP50C and is required to localize the cytokinesis initiation complex.

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring.

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis [1-5] postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA [6], which subsequently recruits contractile-ring components such as actin, myosin, and Anillin [1-3]. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. Here, we report the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. We find that the spindle-associated component, RacGAP50C, which specifies the site of cleavage [1-5], interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring [7-10]. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex.

Centrosomin represses dendrite branching by orienting microtubule nucleation.

Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization.

Dissecting protein interactions during cytokinesis.

Appropriate assembly and constriction of the acto-myosin based contractile ring is essential for the final separation of the two daughter cells in mitosis. This is orchestrated by the small GTPase Rho as well as convergent signals from the prior events of mitosis. Contractile ring assembly requires the physical interaction of structural proteins like the microtubules of the central spindle, motor proteins and Rho activators. These and the interaction of newly localised proteins downstream of active Rho are essential for stability of the contractile ring and its proper constriction. Here, we discuss our recent findings that reveal a complex network of protein interactions during the early stages of cytokinesis. This includes evidence for a direct interaction between Polo Kinase and RacGAP50C as well as unpublished data suggesting other interactions of interest within the contractile ring.

mDia1 targets v-Src to the cell periphery and facilitates cell transformation, tumorigenesis, and invasion.

The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.